Alphaviruses are maintained in nature in a cycle that involves mosquito and vertebrate hosts. Several alphaviruses are significant human pathogens, and are among the primary causes of mosquito-borne viral encephalitis in the Americas. Due to the lack of effective vaccines to many of these viruses, research into novel non-vaccine based antiviral strategies has been initiated. These strategies include the engineering of virus resistance through genetic manipulation of the mosquito vector. To accomplish this goal it will be necessary to characterize virus/host interactions that are required for virus replication in the mosquito, and to devise intervention strategies that target these interactions directly. The broad objective of this proposal is to characterize virus mosquito, and to devise intervention strategies that target these interactions directly. The broad objective of this proposal is to characterize virus/mosquito interactions that may provide targets for intervention in the natural virus maintenance cycle. The specific objective of this application is to identify and characterize the mosquito furin endoprotease, and to investigate the role of this enzyme in alphavirus replication. Furin-mediated cleavage of the viral glycoprotein precursor, PE2, occurs during virus replication in vertebrate and arthropod cells and is required for normal virus maturation processes. Inhibition of PE2 cleavage has been linked to attenuation of viral virulence in a model vertebrate host and to an arthropod cell-specific growth restriction. A furin enzyme (Dfurin1) of Drosophila melanogaster has been characterized, however, no mosquito furin enzyme has been identified or studied directly. The experiments described in this proposal will utilize genetic reagents based on the Durin1 gene to construct and characterize a cDNA clone encoding the mosquito homologue of the furin enzyme. Genetic constructs based on this clone will be used to express and characterize the furin gene product, and to characterize the distribution of mosquito tissues in which the furin gene is transcribed at levels detectable by in situ hybridization. The potential for targeting the PE2/furin interaction a an antiviral strategy will be addressed by investigating the replication, dissemination, and tissue tropism phenotypes of PE2 cleavage-defective Sindbis viruses within living mosquitos, and by investigating the effects that furin specific antisense RNA expression has upon Sindbis virus replication within cultured mosquito cells.